KR102473641B1 - Apparatus and method for temperature monitoring of cryopreserved biological samples - Google Patents

Abstract

The present invention relates to a method for monitoring the temperature of a cryopreserved biological sample. The present invention also relates to a device for temperature monitoring of a cryopreserved biological sample. The temperature monitoring device 10 for a cryopreserved biological sample of the present invention includes a sample container 1 having an accommodation space 2 for accommodating a biological sample 6 . The device of the present invention has at least one chamber (11) whose interior space is not connected to the accommodation space (2) for fluid flow and is partially filled with a label material (7) whose melting point is in the range of -20 to -140 ° C. ). The chamber 11 is such that if the label material 7 is located in a liquid state in the first sub-zone 12a of the chamber, the label material 7 is delayed in the second sub-zone 11 of the chamber 11. It includes a barrier 13 that allows it to reach.

Description

Apparatus and method for temperature monitoring of cryopreserved biological samples

The present invention relates to an instrument for monitoring the temperature of a cryopreserved biological sample. The present invention also relates to a method for monitoring the temperature of a cryopreserved biological sample.

Low-temperature preservation (cryopreservation) of cells is the only way to restart life activity by suspending life activity at the cellular level (while maintaining life) and warming it to physiological temperature. Cryopreservation has evolved over the past decades through biobanks and has become an essential component for hospitals, pharmaceutical companies, species conservation, environmental protection and health provision. The biological material is stored in low-temperature suitable containers (freezing containers) of various sizes, such as tubes, straws and bags. In cryopreservation, the stored biological material is usually frozen at a temperature of -80 ° C or lower while maintaining the vitality of the sample material, and in the case of a living collection, it is frozen at -140 ° C or lower, which is the temperature of liquid nitrogen. In this specification, the term 'frozen sample' means a sample preserved in a frozen state or a sample to be cryopreserved.

A number of techniques have been developed for the cryopreservation of large samples, for example samples such as blood or tissue. Modern pharmaceuticals, genetic engineering and biology tend to extend cryopreservation of microscopic samples. Examples include small suspensions (less than a milliliter) in which suspended cells or populations of cells are suspended. Cryopreservation of cells cultured in a test tube is usually made in a suspension (suspension) state. However, most of the biomedically significant cells require substrate contact for their survival and proper development. Therefore, the sample is frozen while confined to the substrate after incubation.

The quality of cells is critically important because they are used as products for cell therapy, pharmaceuticals, and biotechnology as a national resource. The storage period can range from a few days to several decades, and long-term preservation has become increasingly important. The sample is stored in a cooled container, and is usually located in a metal storage box and rack, and is subjected to temperature changes when the sample is newly stored or removed. In the case of storage of living organisms (cells, cell suspensions and tissue fragments), large temperature rises in deep cooling as well as interruption of frozen distribution, which can have fatal adverse effects on samples, should be avoided. Although it is kept in a frozen state, when used in a medical field, the value of the sample is reduced while the temperature is raised to -80 to -20 ° C, separated from the freezing container, and quality deterioration that can even lead to a life-threatening situation may occur. It can be seen that even if the sample is briefly thawed, the re-frozen condition is not the same as the original condition. However, it is very important not only to confirm the thawing of the biomaterial, but also to record whether the critical temperature in the range of -140 to -20 ° C is exceeded. Temperature control and recording for each sample are required, which has not been satisfied so far, and even so, high-tech instruments are required. When using a frozen sample, if the sample is used after being thawed, even for a short time, an expensive sample can be made worthless and costly.

Therefore, the technical problem to be achieved by the present invention is to provide an improved method for monitoring the temperature of a cryopreserved bio sample, thereby overcoming the problems of the prior art and providing a simplified implementation of the method. In addition, another object of the present invention is to provide a temperature monitoring instrument for cryopreserved biological samples that overcomes the problems of the prior art.

In addition, another object of the present invention is to provide a possibility to check whether a frozen sample has exceeded a predetermined critical temperature even for a short time from a marker as simply as possible. In the present invention, it is possible to fix the critical temperature at a temperature in the range of -20 to 140 ° C before freezing. This should be done clearly and quickly even in the frozen samples of each of the millions of samples so that the biomaterial should not be changed and deep freezing should be performed. In the case of storing multiple samples, when selecting and separating samples, the entire rack is taken out and stored again in the storage container, so there is a risk that the sample may change each time. Should be. The apparatus and method of the present invention should be easy to handle, have low-temperature durability and suitability, and should not consume or minimize energy consumption so that the total cost of storing biological samples in a low-temperature state can be reduced to several euros. The material of the present invention must also meet these requirements. It is also desirable to be able to detect not only the exceeding of the critical temperature to be monitored, but also the measurement of the exceeding time.

The object of the present invention is achieved by an apparatus and method having the technical features of the independent claims. Advantageous embodiments and applications of the invention will become apparent from the dependent claims and will be explained in detail from the following description with reference to the drawings herein.

1 to 6 are structural diagrams of various embodiments of a temperature monitoring device for a cryopreserved biological sample;
7 is a flowchart of an embodiment of a method for temperature monitoring of a cryopreserved biological sample;
8A, 8B and 9A are melting diagrams of liquid mixtures, respectively;
9B is a table summarizing melting points of pure liquids;
10 is a table showing miscibility of solvent matrices;
11 shows another embodiment of the temperature monitoring mechanism of the present invention;
12 shows another embodiment of the temperature monitoring mechanism of the present invention.

According to a first aspect of the present invention, the above object is achieved by a device for temperature monitoring of a cryopreserved biological sample. The device includes a sample container having an accommodation space (sample reservoir) for accommodating a biological sample. The sample vessel is in particular a cryogenic sample vessel. The receiving space contains a cryopreserved sample.

The device further comprises at least one chamber having an interior space that is not fluidly connected to the accommodation space and partially filled with a label material having a melting point in the range of -20 to -140° C. at atmospheric pressure, i.e., 1013.25 mbar. do. The melting point is preferably in the range of -20 to -100 °C. In this case, if the labeling substance is located in the liquid phase in the first subzone of the chamber, the chamber preferably comprises a barrier which delays, for example temporally, the arrival of the labeling substance in the second subzone. The barrier is impermeable to the label when it is frozen. Thus, the barrier serves as a retardant configured in such a way as to retard the flow of the liquid phase from the first subzone to the second subzone of the chamber.

As a result of the chamber of the device according to the present invention, an additional configuration is provided which can be used as a labeling element or a labeling device partially filled with a labeling material so as to exhibit an undesirable exceeding of the critical temperature. If the critical temperature to be monitored corresponding to the melting point of the label material is exceeded or if the critical temperature is higher than the melting point and the viscosity of the liquefied label material is sufficiently reduced, the label material is liquefied and the first sub-chamber of the chamber is liquefied. From the zone it flows into the second sub zone. Therefore, if at least a partial labeling material is located in the second sub-zone upon confirmation later, it can be concluded that the critical temperature is exceeded in the middle even for a very short time.

An advantage of the device according to the present invention is that, thanks to the barrier, even if the label substance is liquefied, it does not immediately flow completely into the second sub-zone of the chamber, but rather takes a certain amount of time and is delayed. Thus, it is possible to infer a measure of the duration of time above the melting point from the amount of the labeled substance located in the second sub-zone at a specific time. In this case, the correlation between the amount of the labeling substance and the duration above the melting point can be determined experimentally in advance, for example, by providing the chamber wall in the form of a scale. The graduations can be plotted corresponding to corresponding durations above the melting point, for example as a function of the degree of filling of the labeling substance in one of the sub-zones or as a function of the length of the diffusion section in the second sub-zone.

According to one embodiment of the invention, the wall of the second sub-zone of the chamber and/or the wall of the first sub-zone of the chamber may include a scale indicating the duration of the temperature above the melting point.

According to another aspect of the present invention, the walls of the second sub-zone and/or the walls of the first sub-zone of the chamber may be transparent or translucent at least at one point. As a result, it is possible to more easily determine whether the label material is located in the second sub-zone and/or the degree of filling of at least one of the two sub-zones. Also, the entire chamber may be made of a transparent or translucent material. That is, the chamber can be viewed from the outside.

For improved detection, the labeling material may further include a labeling additive capable of increasing the sensitivity of physical properties of the labeling material. The labeling additive may be, for example, a dye, and allows the labeling material to be colored or dyed, ie non-transparent, so that its presence is more clearly visible optically in the second sub-zone.

In principle, the dye is not particularly limited as long as it satisfies the following conditions:

-Those that produce a strong dyeing effect even when used in small amounts or at low concentrations (for example, adding less than 1% by volume starting from a saturated salt solution, generally using one thousandth or less)

- cold resistance

-Light fastness at dispatch temperature and moderately low temperature

- Soluble in components of the labeling substance

-Do not separate during freezing

-Do not react in case of contact between label material and plastic material

The dye is preferably selected from the group comprising triphenylmethane dyes, rhodamine dyes, in particular xanthine, azo dyes as well as phenazine and phenothiazine dyes.

In a more particular embodiment, the dye may be selected from the group consisting of oil red, methyl red, brilliant green, rhodamine B, neutral red, and methylene blue, or may be a cell dye used in cytology.

The labeling additive may be particulate, in particular, nanoparticles capable of enhancing scattering behavior and/or polarization when the labeling material is irradiated with an electromagnetic field. As a result, it is possible to more reliably measure the liquid level in the second subzone by means of optical measurement, scattering measurement and/or polarization measurement. The labeling additive may be in a conductive position. The conductivity or resistance of the label can be influenced by the addition of conductive particles. In this way, the presence of a marker can be detected by measuring conductivity or resistance. In order to more reliably determine the presence and/or level of the labeling material in the first sub-zone and/or the second sub-zone as a result of the added labeling additive, the corresponding physical properties of the labeling material are measured with a conveniently configured measuring device. can be detected.

A sample container is a container suitable for cryopreservation, such as a tube, a straw (called a seed tube), a bag for blood or stem cells, a box, or other container suitable for cryopreservation. Such containers are referred to as cryogenic tubes, cryogenic straws, cryogenic bags, cryogenic boxes or generally cryogenic containers.

Cryogenic tubes are also called biobank or cryobank tubes. The cryogenic tube includes a receiving part forming an inner space for accommodating a biological sample. The cryogenic tube generally further includes a cover capable of closing the accommodation space. The cover includes a coupling part that can be rotated using a tool. The cryogenic tube may further comprise a substrate element comprising a marking in the form of a machine readable code.

A substance having a melting point corresponding to a predetermined critical temperature to be monitored for excess can be selected as the labeling substance. The labeling material is a liquid or liquid mixture, and has a melting point corresponding to a designed critical temperature. For example, a mixture of water (H2O) and ethanol (C2H6O), a mixture of water (H2O) and potassium hydroxide (KOH), or a mixture of water and antifreeze may be selected as the labeling material. The mixing ratio is adjusted according to each melting diagram representing the melting point profile as a function of the mixing ratio so that the melting point of the liquid mixture has the desired value, i.e., the critical temperature to be monitored.

According to a preferred embodiment of the present invention, the labeling material is octan-1-ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane -1,3-diol, butan-2-ol, pentan-1,5-diol, pentan-1-ol, cyclopentanol, and at least one alcohol selected from the group consisting of benzyl alcohol. More preferably, the alcohol is selected from propane-1,3-diol, propan-1,2-diol and butan-2-ol.

According to another preferred embodiment of the present invention, the label comprises at least two different alcohol components:

a) Octan-1-ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, butane-2- one alcohol selected from the group consisting of ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, and benzyl alcohol;

b) Octan-1-ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, butane-2- It is an alcohol selected from the group consisting of ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, and benzyl alcohol, which has a lower melting point than the alcohol of component a),

The mixing ratio of components a) and b) is adjusted so that the melting point of the mixture lies at a temperature in the range of -20 to -160°C, more preferably in the range of -25 to -160°C or -50 to -150°C.

A more specific embodiment is characterized in that the label material comprises one of the following combinations of components a) and b):

-Mixing ratio of 5 to 95% by volume of octan-1-ol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of octan-1-ol and pentan-1-ol

-Mixing ratio of 5 to 95% by volume of octan-1-ol and propan-1,2-diol

-Mixing ratio of 5 to 95% by volume of nonan-1-ol and butan-2-ol

- Mixing ratio of 5 to 95% by volume of nonan-1-ol and propane-1,2-diol

-Mixing ratio of 5 to 95% by volume of nonan-1-ol and pentan-1-ol

-Mixing ratio of 5 to 95% by volume of propane-1,2-diol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of propane-1,2-diol and propane-1,3-diol

-Mixing ratio of 5 to 95% by volume of propane-1,2-diol and butane-1,2-diol

-Mixing ratio of 5 to 95% by volume of propane-1,3-diol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of propane-1,3-diol and butane-1,2-diol

-Mixing ratio of 5 to 95% by volume of pentane-1,5-diol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of benzyl alcohol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of pentan-1-ol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of pentan-1-ol and methanol

-Mixing ratio of 5 to 95% by volume of cyclopentanol and butan-2-ol

-Mixing ratio of 5 to 95% by volume of cyclopentanol and propane-1,2-diol

-Mixing ratio of 5 to 95% by volume of cyclopentanol and pentan-1-ol

- a mixing ratio of 5 to 95% by volume of cyclopentanol and butane-1,2-diol;

The value of the mixing ratio indicates the ratio of the previously described components in the total mixture of both components in each case.

According to a more preferred embodiment of the present invention, an example of the label mixture is propane-1,2-diol and butan-2-ol (having a melting point of about -90 ° C) having a mixing ratio of 40 to 60% by volume, Propane-1,2-diol and propan-1,3-diol having a mixing ratio of 30 to 70% by volume or propan-1,3-diol and butan-2-ol having a mixing ratio of 30 to 70% by volume can

In addition, the labeling material preferably includes at least one of the aforementioned dyes in addition to at least one alcohol. The dye is particularly preferably at least one selected from the group including Oil Red, Methyl Red, Brilliant Green and Rhodamine B.

In a more preferred embodiment of the present invention, the labeling material is at least one dye selected from the group including Oil Red, Methyl Red, Brilliant Green, and Rhodamine B, as well as propane-1,3-diol, propane-1, It is characterized by containing two alcohols a) and b) selected from 2-diol and butan-2ol.

The concentration of the dye in the alcohol can vary greatly depending on the type of dye and alcohol.

For dark dyes, the concentration should be kept as low as possible so that the dye molecules do not change the freezing and thawing properties of the alcohol in which they are dissolved or increase the viscosity. The concentration of the dye is generally less than 10% by volume, more preferably less than 1% or 0.1% by volume, ie in the range of parts per thousand or less.

In one variant of the present invention, the critical temperature to be monitored does not directly correspond to the melting point of the labeling substance, but is a temperature above the melting point at which the viscosity of the molten substance is lowered to the extent that the required liquid phase movement can occur. corresponds to

This temperature is also referred to as the critical temperature in the present invention, and is usually a temperature in the range of 3-30°C or 5-30°C above the nominal melting point, such as 3-10°C, 3-20°C, 5-10°C. or in the range of 5-20° C. or higher.

In an embodiment of the present invention, the labeling material is in the range of 10 to 10 ⁶ mPa*s, more preferably 10 to 10 ⁴ mPa*s, when the liquid mixture at a temperature in the range of 3-30°C or 5-30°C or more above the melting point. It is characterized by having a viscosity in the range.

According to a preferred embodiment of the present invention, the barrier is disposed in the second sub-zone, is adjacent to the first sub-zone, and may be made of a material having a liquid absorption structure. The material may be a porous material or medium. The material may be, for example, an absorbent, for example filter paper, i.e. common kitchen towels or cigarette filter paper, compacts, cellulose discs, i.e. tissue cellulose discs, plaster and/or chalk dust, porous membranes, cloth or It may be a knitted fabric, a layer of nano or microporous aluminum oxide. This can be influenced by its physical properties, such as a change in porous structure, and can promote absorption of liquid by capillary force. It may be any other material suitable for absorbing the liquid in other ways. According to an embodiment of the present invention, the label material initially exists in a frozen state on the barrier made of a liquid absorbent structure material and cannot penetrate the barrier. After exceeding the liquid phase, i.e., the melting point of the label material, the label material located in the first sub-zone slowly diffuses into the material adjacent to the first sub-zone and having a liquid-absorbing structure. As a result, the label material permeates the material. The label material reaches the second subzone of the chamber in this delayed manner. The degree of delay depends on the diffusion rate.

Another advantage of the present invention is that the location of the diffusion front can measure how long the sample inside the accommodation space has been exposed to an unacceptable temperature range according to the labeling material. This can be determined by determining the location of the diffusion front. The rate of diffusion is itself a function of viscosity as a function of temperature; However, between a few minutes and a few hours to a few days is often sufficient to perform the evaluation.

Therefore, it is possible to detect the exceeding of the critical temperature as well as its duration. The basic principle of this embodiment is to provide a closed space containing a frozen label substance that is solid at storage temperature but becomes liquid when its melting point is exceeded in the sample container or on a material in which the label material can slowly diffuse on the sample container. .

Combinations of multiple liquids with different melting points and multiple diffusion zones, combined or separated, are also possible, for a more precise determination of the time in relation to the duration of the overtemperature.

According to one aspect of the present invention, in particular, for the activation of temperature monitoring, the labeling material is contained in the receptor tightly surrounding the labeling material in liquid state, at least until the receptor is damaged. The temperature monitoring mechanism may further include an activating part which is movably guided relative to the receptor and which is movable from a first position, referred to herein as a starting position, to a second position, also referred to as an activating position. Movement to the activation site causes the active part to break the receptor at least at one point in such a way that the mechanical pressure causes the receptor to permeate the label material in liquid state. That is, a mechanism is provided to cause the temperature monitoring mechanism to switch to an activation or activation mode at a predetermined point in time. The potential for activation facilitates trade and stock management because the liquid label material cannot penetrate adjacent materials having a liquid permeable structure until activated.

The receptor can be specifically embodied in the form of a plastic cushion that can be manufactured at low cost. In addition, the receptor may be embodied in the form of a glass bead capable of generating an audible sound that breaks when broken, enabling acoustic feedback of activation.

The sample container may further include a cover for closing the receiving space, and the cover may be integrated with at least one chamber. In this case, the cover further includes a base portion that can be coupled to the sample container by pressing and/or screwing, and in a state in which the screw is coupled, the accommodation space for accommodating the biological sample is closed. The base may include an H-shaped cross section. To form the at least one chamber, the base may further include a groove in which a receptor having a label material and a material having a liquid absorbent structure is arranged. As described above, the temperature monitoring mechanism may further include an activating part that is movably guided with respect to the receptor. The active part can be threaded onto the base. The activating part may further include a protruding part protruding from the activating part toward the receptor, for example, in the form of a tip or a spine.

Another embodiment according to the present invention provides that at least one chamber of the device is integrated into the base of the sample container. In this case, in order to form at least one chamber, the base region of the sample container includes a groove in which a receptor having a labeling material and a material having a liquid absorbent structure is embedded. The groove may be closed by a base in which the activating part is movably guided. The base may be embodied in the form of a plastic cap. Preferably, the base comprises a mechanically and/or optoelectronically readable code, in particular a barcode, 2D code, 2D barcode and/or QR code.

In a variant of the invention, the active position is fixed by means of a rest position formed by the base, up to which position the active part can be pushed into the base. Furthermore, the base may be fixedly connected to the base region of the sample container, and in particular may be fixed to the base region of the sample container by bonding, melting, welding or other fixing means. As a result, the tamper-proofing of the temperature monitoring is improved.

In an advantageous variant of the invention, the surface of the material adjacent to the first subzone comprises a covering. When the device of the present invention is cooled to a storage temperature below the melting point of the labeling material, the covering irreversibly converts from a first covering state impervious to the labeling material to a second covering state permeable to the labeling material. It is built to experience. In the first state, the covering is impervious to the liquid labeling material, that is, non-porous. Thus, the covering forms a separator. The covering may be, for example, a membrane, a covering film, a skin, or a film or similar formed to correspond, and when cooled to a storage temperature, it shrinks and is torn or becomes a liquid-permeable porous material so that the liquid labeling material is formed through at least one crack. It is allowed to flow from the first sub-zone to the second sub-zone through an opening formed by a point or general entry point.

This manufacturing principle has the advantage that the chambers already filled with the labeling material can be manufactured in advance and made into a closed form. These chambers can be made and stored in ready-made form and cooled with the sample in the sample container.

Thus, these chambers used as labeling devices can be stored in preloaded and filled form at room temperature for as long as desired because the covering is impermeable.

The material of the covering should have greater shrinkage than the chamber material when the temperature decreases, and as a result, it is preferable to form an opening so that the liquid labeling material can pass through the material having liquid absorbency. In the case of correct storage, despite the presence of the opening, the label material freezes and therefore does not penetrate into the material or flow into the second sub-zone of the chamber.

According to another variant of the invention, the covering, for example in the form of a membrane, can be converted into a permeable second state by mechanical bending or press-fitting. The covering can thus be made permeable to the liquid label material by bending or press-fitting only immediately before use.

In another advantageous variant of this configuration, the structure and/or composition of the material with the liquid-absorbing structure is such that the diffusion rate of the labeling substance in the material decreases nonlinearly with increasing distance from the first subzone. can

In this way, only a very short time (seconds to minutes) is consumed when the temperature exceeds in the upper zone of the material, i.e. the part of the material facing the first sub-zone, and in the lower zone, i.e. the part of the material facing away from the first sub-zone. It may prove to consume a very long period (hours to days).

An advantage of an embodiment of the present invention is that the barrier is a separating element that is permeable with respect to the liquid label material and is disposed between the first and second subzones. Thus, the barrier forms a separation membrane between the first and second sub-zones, and is permeable to the labeling material in a liquid state but not to the labeling material in a frozen state. The separation element permeable to the liquid labeling material may be embodied in the form of a porous separation wall, membrane, film, skin or capillary system.

In this case, the feasibility according to the present invention is that if the device is cooled to a storage temperature below the melting point of the labeling material, the separating element ruptures at least one point as a result of thermal contraction, and as a result, the labeling material ruptures at least one point. It is provided so that it can flow in a liquid state from the first sub-zone to the second sub-zone through the opening formed by the branch.

This variant has the advantage that since the separating element is impermeable, such a chamber acting as a labeling device can be pre-fabricated, pre-filled and stored at room temperature for as long as desired. If desired, the chamber may be placed in a sample container and cooled together with the sample placed in the sample container, and at least one rupture point is soon formed. In the case of correct storage, the label material is cooled and therefore cannot pass through the material or flow into the second sub-zone of the chamber despite the opening.

The release element may include a predetermined rupture point, which is a location at which the release element ruptures while the device is cooled from storage temperature. The above predetermined point of rupture refers to a point of the separation element at which the separation element ruptures during cooling at storage temperature. This has the advantage that the position and size of the rupture point are predetermined and, therefore, the perfusion rate of the labeling material is determined as a result when the labeling material is in liquid form.

The predetermined rupture point may be embodied, for example, by a thin incision at a specific point of the separation element, for example, in the form of a point-shaped thin incision at the edge of the separator or in the form of a cross-shaped line on the separator. Such a separator may be manufactured using, for example, an injection molding process. Alternatively, the material may also be formed in an irregular manner.

Another advantage of the variant of the invention with the separation element is the presence of gas in the second sub-zone. Instead of a gas, a substance having a lower melting point than the labeling substance may be included in the second sub-zone. The material may be a material that is liquid at storage temperature or at least prior to liquefaction of the labeling material.

The non-applied temperature rise time may be determined based on changes in physical properties of the gas or material, for example, color, transparency, optical rotation angle, impedance, etc. due to mixing with the label material flowing into the second sub-zone. can Since this process is irreversibly formed in the connected space, a temperature monitoring device formed in this way can be considered non-operable.

In order to fill the first sub-area of the chamber with the label material, it is possible in the context of the present invention that the outer wall of the chamber comprises an openable opening in the first sub-area. The opening may be closed by welding, other material, or other sealing method after filling with the labeling material.

At least one chamber in the device of the present invention may be formed by a vessel having one or more cavities disposed and/or displaceable outside the sample vessel. As used herein, the term "disposed and/or displaceable" includes the meanings of "coupled and/or connectable", "coupled and/or matable", and "connected and/or connectable". . Accordingly, a vessel for forming at least one chamber partially filled with a label material can be distinguished from a sample vessel.

In this case, the container may be placed and/or placed outside or inside the container. A possibility of an embodiment according to the present invention is that the vessel for forming the at least one chamber is detachably coupled to the sample vessel. The detachable attachment includes a slide or press method specifically on the sample container. This has the advantage that the container can be stored or prepared (eg, filled with a labeling material) spatially separated from the sample container.

For example, the sample container may include a cover for closing the receiving space. According to a preferred embodiment of the invention, the at least one chamber can be integrated into the cover, eg the head and/or shaft of the cover. For example, the cover may include a shaft coupled to an upper section of the receiving space of the sample container, and at least one chamber may be integrated with the shaft.

The arrangement of the at least one chamber within the cover has the advantage that no additional installation space is required outside the sample container. Another advantage is that at least one chamber capable of serving as a labeling device can be spatially separated from the rest of the sample container, stored with a cover, and prepared (eg, filling the chamber with a labeling material and freezing). . Integrating the chamber with the shaft of the cover is particularly advantageous. According to this variant, the shaft of the cover comprises a cavity partially filled with an indicia material. In particular, in the case of a cryogenic tube, often only the lower subspace of the accommodation space is filled with the biosample, and as a result, the upper subspace can be used for the placement of the label material.

Another realization possibility according to the present invention is that the at least one chamber is formed by a container and a receptor coupled to the outer wall of the sample container, for example a sleeve or insertion pocket inserted or capable of being inserted to stay on the sample container. to provide.

According to another embodiment of the present invention, the at least one chamber may be formed by a push-on structure of a double wall structure capable of being pressed or slid on the outer surface of the sample container, and at least around the sample container in a compressed state. partially combined This variant is particularly advantageous for cylindrical sample containers, in particular for cryogenic tubes. The push-on structure of the double wall structure may be embodied in the form of a hollow cylinder or a partial hollow cylinder in which the inner diameter corresponds to the outer diameter of the sample container so that the push-on structure is coupled around the sample container in the form of a cylinder in a cuff or crimp manner. can

Furthermore, the sample container may be fixed to the push-on structure by bonding, melting, or other fixing methods. As a result, removal of the push-on structure for the purpose of manipulation, for example replacing a push-on structure exhibiting inadequate heating with a new push-on structure, can be avoided.

However, the sample container may be a known bag for storage and/or cryopreservation of blood or stem cells coupled to the cassette. At least one chamber may be formed by a container coupled to an outer surface of a bag coupled to the cassette. At least one chamber may be formed by a container floating freely inside the bag. In this case, the container with the label material can be adjusted in terms of density so that it comes to the center in the liquid phase of the bag and the temperature of the center of the bag can be measured.

According to another preferred embodiment of the present invention, the device may include a plurality of chambers and the aforementioned barrier, each partially filled with a label material having a melting point in the range of -20 to -140°C. For example, in order to form a plurality of chambers, the container may include a plurality of sub-cavities formed by partition walls. In this case, each sub-cavity forms a chamber containing a labeling material and a barrier. Label substances in the chamber may have different melting points. Accordingly, various temperature thresholds can be monitored, and the labeling substances or mixtures thereof are selected and/or mixed ratios are adjusted so that each corresponds to a selected one of the temperature thresholds to be monitored. This embodiment can precisely limit the temperature interval that the sample must reach.

According to another embodiment of the present invention, at least one chamber is integrated with the sample container itself, that is, the sample container itself may include one chamber or a plurality of chambers therein. As a result, a separate element disposed outside the sample container for forming the at least one chamber can be omitted. For example, the accommodation space of the sample container for forming the at least one chamber may be embodied in a double-walled structure having an inner wall and an outer wall, and an intermediate space between the inner wall and the outer wall may be partially filled with a label material. For the multi-chamber type, the intermediate space can be divided into sub-spaces by dividing walls.

According to a preferred embodiment of the present invention, the device of the present invention is capable of detecting the presence of a label substance in the second sub-zone of the chamber and/or the level of the label substance in the first sub-zone and/or the second sub-zone of the chamber. It may include a measuring mechanism formed to be able to. The measuring instrument may be an optical or optical-electrical measuring instrument for confirming the shape change of the labeling substance, for example, measuring optical transmission, light scattering or light reflection.

According to a second aspect of the present invention, the above object is achieved by a method for monitoring the temperature of a cryopreserved biological sample using the temperature monitoring device described above. For the avoidance of repetition, variations of examples, especially advantageous embodiments, relating to the apparatus should be regarded as having been disclosed in accordance with the method of the present invention and may be claimed as such.

A substance having a melting point corresponding to a predetermined critical temperature to be monitored, the excess of which is to be monitored, may be selected as the labeling substance.

According to the method of the present invention, there is provided the temperature monitoring device described herein above, wherein the device includes at least one label substance in a frozen state in a first sub-zone of the chamber. The receiving space of the sample container contains a cryopreserved biological sample. The method of the present invention includes cryopreservation of instruments for cryopreservation. The method of the present invention also includes determining whether the labeling substance is located in the second sub-zone of the chamber at a later time point.

If this is the case, it can be concluded that the melting point of the labeling substance is exceeded, and therefore the critical temperature to be monitored is also exceeded, especially when the excess is only released for a short time.

A particular advantage of the present invention is that the presence of the labeling substance in the second sub-zone directly indicates that the frozen sample has been heated above a definable critical temperature, no matter how short the time. This can be easily and quickly confirmed by visual inspection as well as by a device configured to measure it without taking or removing the sample from the sample container.

According to another advantageous embodiment of the method of the invention, a parameter indicative of a measure of the amount of labeling substance that has moved into the second sub-zone of the chamber and/or is located in the first sub-zone can be determined. These parameters represent a measure of the time a sample remains at a temperature above its critical temperature.

The parameter is, for example, a variable representing the amount of the labeling substance in the first sub-zone or the second sub-zone. The parameter may be the filling level in the second sub-zone or the length of the diffusion section reached by the label material in the material with the liquid-absorbing structure.

According to an embodiment of the method of the present invention, a temperature monitoring device is used to carry out the method, the device comprising a separating element or covering which becomes permeable to the liquid label material during cooling. Upon cooling of the device to a storage temperature below the melting point of the labeling material, the separation element disposed between the first sub-zone and the second sub-zone ruptures at least at one point as a result of heat shrinkage so that the liquid labeling material is at least one ruptured point. As described above, it is possible to flow from the first sub-zone to the second sub-zone through the opening formed by

In addition, when the device is cooled to a storage temperature below the melting point of the labeling material, the surface of the material adjacent to the first sub-water area includes a covering such as a membrane or a covering layer and is impermeable to the labeling material in the first covering state. It is also configured to be converted to the second covering state that is permeable to the label material as described above.

According to an embodiment of the method of the present invention, at least one of the following test steps may be performed to confirm the functionality of the temperature monitoring device, in particular the functional capability of the indicator device.

First, after the labeling material is filled in the first subzone before the labeling material is cooled, it may be tested whether or not the labeling material is present in the second subzone of at least one chamber. In this state, the separation element acting as a barrier is still intact and consequently no label material is present in the second sub-zone below the first sub-zone. However, if this happens, the chamber cannot be used because it has already been damaged. The same judgment applies if a chamber with a covering is used instead of a chamber with a separation element.

Second, it is possible to check whether there is no label material in the second sub-zone of the at least one chamber after cooling the label material located in the first sub-zone of the at least one chamber and before cold storage of the device for cryopreservation. After cooling the device to a temperature below the melting point of the labeling material or storage temperature at which the separation element should rupture, the separation element should rupture and the labeling material should freeze so that the labeling material does not exist in the lower second sub-zone. If not, a new chamber must be used. The same judgment applies if a chamber with a covering is used instead of a chamber with a separation element.

Thirdly, if the frozen sample is removed for use and there is no labeling material in the second subzone of the chamber, the covering has transitioned to a second state of permeability or at least one of the isolation elements of the chamber or the covering has properly ruptured. You can check whether it has been done or not. If the separation element or covering does not rupture, it can be seen that this does not indicate that the melting point of the labeling substance has been exceeded. This can be verified, for example, by placing a suitable chamber with a labeling material for a predetermined period of time after thawing. The sample may be processed in other steps during this time. If the separation element or covering is properly ruptured and the storage temperature is maintained below the melting point of the label material during the entire time, the liquid label material at room temperature will pass through the barrier and migrate into the second sub-zone.

All these test steps are carried out within the framework of temperature monitoring of cryopreserved samples. In this way, the functional capability of the chamber can be demonstrated and it can be clearly shown that the storage temperature meets the requirements over the entire time period.

In the present specification, the term 'sample container' refers to a container specially designed for cryopreservation. The sample container is preferably made of low temperature compatible plastic material suitable for temperatures below -140 °C. The plastic material must be durable without deformation or damage even in repeated temperature changes. Preferably, the plastic material has an absorption property of less than 1%, more preferably less than 0.1%, based on the total weight. The cryopreservation material according to the invention is for example based on polyurethane or polyethylene.

As used herein, the term 'biological sample' refers to a biological material, such as a cell, tissue, cell component, biological macromolecule, etc., which is to be frozen and preserved in a sample container, and is suspended and / or combined with a base material can be used The substrate may be formed to attach and accommodate cells, which are part of a biological sample, and may be disposed in the receiving space.

Preferred embodiments of the present invention described above can be implemented in a mutually coupled form. Hereinafter, a detailed description of the present invention and its advantages will be described with reference to the accompanying drawings.

1 to 4 are structural diagrams of various embodiments of a temperature monitoring device for a cryopreserved biological sample;

5 shows the structure of a variant of the chamber with a separating element as a barrier;

6 is a flowchart of an embodiment of a method for temperature monitoring of a cryopreserved biological sample;

7A, 7B and 8A are melting diagrams of liquid mixtures, respectively;

8B is a table summarizing melting points of pure liquids;

9 is a table showing miscibility of solvent matrices;

10 shows one embodiment of the temperature monitoring mechanism of the present invention;

11 shows another embodiment of the temperature monitoring mechanism of the present invention;

12 shows another embodiment of the temperature monitoring mechanism of the present invention.

Elements that play the same or equivalent role in each figure are indicated by the same reference numerals or are not partially separately described.

1A shows a sample container in the form of a cryogenic tube (tube) 1 and is representative of other cryogenic sample containers, such as straws, bags, boxes, and the like.

The cryogenic tube includes a receiving space 2 for a bio sample in which a bio material is placed. Here, the biosample is the cell suspension 6. The cryogenic tube shown in FIG. 1A is still without a screw cover lid. The cryogenic tube shown in FIG. 1B further includes a cover 3 for closing the container, and includes a coupling part 4 capable of rotating the cover 3 using a tool (not shown) on the top in case of automation. Cryogenic tube 1 may include a base 5 into which a bar code or other mark is optionally inserted. In this configuration, the cryogenic tube 1 is placed vertically in the reservoir and stored in the cold reservoir.

A system ready for storage is shown in FIG. 1B. The cover includes a head portion placed on the accommodating space 2 and a shaft 5 formed in the head portion and coupled to the accommodating space 2 with screws. A thread 8 is formed in the corresponding space of the accommodating space 2 . The chamber 11 forming the enclosed space 12 is integrated with the screw cover 3. The inner space 12 of the chamber 11 is represented by black and dots in FIG. 1B.

Located in the space 12 in the second sub-space 12b is a small cavity (first sub-zone) 12a thereon filled with a porous or liquid-absorbing material 13 and a label material through an introductory opening 14. When cooled below a predetermined critical temperature, for example -70°C, the labeling substance 7 is frozen, and when it exceeds this temperature, it is liquefied again.

Through the selection of appropriate liquids and mixture ratios of liquids, their melting points can be set to desired values in the range of -20 to -140 ° C, and appropriate labeling substances 7 can be selected according to the critical temperature to be monitored, FIG. 6 It will be explained in detail below along with 9 to 9.

Immediately prior to setting the storage temperature of the cryogenic tube 1 below, for example -140°C, the filling of the first subzone 12a is expediently carried out during cooling, sufficient time for the label material 7 to permeate the material 13; There should be no time, which could be minutes, hours or days depending on the design.

At storage temperature, labeling material 7 becomes a solid phase and no further changes to the batch as shown in FIG. 1B occur. The opening 14 is welded closed or sealed with material 15.

An apparatus 10 formed in this way consisting of a chamber 11 and an integrated sample container 1 is configured for temperature monitoring of a biological sample 6 stored in a freezer. Figure 1B shows the instrument without exceeding the melting point of label 7 at the beginning and middle. Figure 1C shows the mechanism of the state when the melting point of the labeling substance 7 is exceeded in the middle.

Thus, if sample 6 is heated at some point beyond the melting point of label material 7, e.g., a dyed alcohol/water mixture having a melting point of -70°C, the now liquid label material permeates material 13, The result is the state of instrument 10 as shown in FIG. 1C. Fig. 1C shows an ecology in which the label material has already spread to the upper zone 13a by the length Δx of the material 13 and has not yet reached the lower zone 13b.

The length (diffusion zone) Δx measures the period of time that sample 6 spends at a temperature above the critical temperature. Since this process is not reversible, unless the entire screw cover 3 is replaced, the system is protected and prevented from being tampered with, which is detected by means of markings, codes or the like.

The structure of this chamber 11 can be modified in various ways. For example, the porous material 13 may be covered by a covering (not shown) such as, for example, a thin skin or membrane, which upon cooling to storage temperature may rupture upon shrinkage or become permeable to the liquid label material. can This construction principle is such that the pre-filled cover is prefabricated and stored in a closed manner, so that when in use the conventional cover can be combined with sample 6 and cooled.

When the temperature decreases, a material that shrinks more than the cover material and forms an opening through which the liquid labeling material 7 can permeate can be suitably used for this covering. Upon correct storage, the labeling material 7 will freeze and will not pass through the material 13 or the second subzone 12b despite the presence of the opening.

Through the structure and design of material 13, various deformation possibilities are revealed. These materials can be influenced in terms of their properties so that they promote liquid uptake as a result of capillary forces, or they can be set such that the desired rate of diffusion decreases non-linearly with distance in a familiar downward fashion. In this way, it can be seen that a very short time (seconds to minutes) is consumed in the upper zone of material 13, whereas a very long time (hours to days) is consumed in the lower zone.

An intermediate layer that can be made permeable from the outside by bending or press-fitting immediately before use can also be used as a covering.

2A, B and C show another configuration of the present invention, and FIG. 2C shows a temperature monitoring device for a cryopreserved biological sample placed in a state after the melting point of the labeling substance 7 is exceeded midway.

Figure 2A shows a cryogenic tube 1 completely closed by a cover 3 normally used as a cryobank. FIG. 1B shows a double-walled push-on part 21 made of plastic and placed on the outer surface of the cryogenic tube 1 as seen in FIG. 1C.

One or more spaces 22 containing therein the label material 7, each located in the upper sub-zone 22a in a frozen state on the porous media 23, are located within these plastic parts 21 in a vertical arrangement in a manner similar to the embodiment shown in FIG. . The porous media is located in the lower subregion 22b of space 22 . Accordingly, the plastic part 21 forms a chamber according to the present invention, or a plurality of chambers when the inner space 22 of the plastic part is divided into a plurality of sub-spaces each containing a label material and a porous medium by a separating wall.

In a manner similar to the embodiment shown in FIG. 1 , when the melting point of the labeling material 7 is exceeded and the labeling material melts, the labeling material 7 diffuses into the material 13 positioned below it.

2C shows a state in which the labeling substance has already diffused into the upper zone 23a of the porous media 23.

For this embodiment, the diffusion zone ranges from a few millimeters to 10 centimeters depending on the size and shape of the sample container. The plastic part 21 may be equipped with a scale 24 to increase the accuracy of timekeeping.

Another embodiment could be a sleeve or cylinder part compressible to the cryogenic tube 1 in a similar manner. To prevent tampering, the attachment portion may be fixed by gluing, melting or other methods.

3A, B and C show another configuration of the present invention, wherein FIG. 3C shows a temperature monitoring device 30 of a cryopreserved biological sample in a state after the melting point of the labeling substance 7 has been midway exceeded.

3A shows cryogenic tube 1 completely closed by cover 3. Unlike the cryogenic tube shown in FIG. 2A, the receiving part 34 into which the container 31 shown in FIG. 3B is press-fitted onto the outer surface of the side of the cryogenic tube 1. This vessel 31 forms an interior space 32 of cylindrical or other shape. The container is prevented from falling out of the receptacle 34 via the disk 37. A labeling material 7 is located in the first sub-zone 32a of the inner space 32, and optionally a separator (not shown) that becomes permeable to the labeling material in a liquid state when cooled to a storage temperature, and a second sub-section of a porous absorbent material 33 below it. Area 32b is located. In the case of porous materials with strong capillary action, the location of the unit plays a dependent role. However, vertical arrangement and storage of samples are recommended.

FIG. 3C shows a temperature monitoring device formed of a cryogenic tube and container 31 in a state after the melting point of label 7 has been midway exceeded. In contrast to the state of the container shown in FIG. 3B, it is evident that the labeled material has diffused into the porous material 33. Δx again represents the length of the diffusion zone.

4 shows a bag 41 for storage of stem cells and blood samples. These bags are often used mounted within a cassette 42 made of aluminum. A temperature monitoring device 40 for a cryopreserved biological sample may be formed from a sample container in the form of a bag 41 and a temperature sensitive system. The temperature sensitive system may be formed of containers 44, 45 or 46 partially filled with label material 7 in a manner similar to the configuration of chambers or containers 11, 21, 31 of FIGS. 1-3. If the label material 7 is in liquid phase in the first sub-zone of the chamber, the container further comprises a porous material 43 through which the label material 7 can flow in a delayed manner to the second sub-zone of the container.

Thus, the container 43 can fit on the outer side of the bag 41. Container 45 may also fit outside or inside cassette 42 . The container can also be fitted either free floating or fixed inside the bag 41 . The modification on the inside requires sterilization and the outside is required to be made of an inert material. Such a system can be calibrated in terms of density to allow the liquid to float in the center of the bag and record the temperature of the center of the bag.

5 shows various embodiments of a chamber or vessel configured as an indicator device for temperature monitoring.

5A shows a vessel 51 whose interior space 52 forms two sub-volumes 52a and 52b separated by a barrier 54. A label material (not shown) is contained within the upper subspace 52a. The barrier 54 may be a membrane or film having a predetermined rupture point, which is indicated by the dashed line 55a and is the point at which the barrier 54 ruptures upon cooling to storage temperature. The barrier is formed to rupture only at a temperature below the melting point of the label material located in the subspace 52a. In this way, the first upper subspace 52 is filled with a label material and then cooled sequentially.

Barrier 54 may be a material that has greater shrinkage properties on cooling than the cylinder material and therefore ruptures to a more or less irregular extent.

A container 51 filled with labeling material may be placed on top of a sample container in which a frozen sample is stored therein, together cooled to a storage temperature lower than the melting point of the labeling material.

In the case of cooling, the barrier is ruptured so that the label material in liquid state located in the sub-volume 52a can flow slowly from the first sub-volume 52a to the second sub-volume 52b through the opening 55b formed by the at least one rupture point.

5B and 5C show embodiments of barriers 54b and 54c with rupture points 56a and 57a, respectively. In each case, FIGS. 5B and 5C show the intact barrier at the predetermined rupture point before cooling on the left, and ruptured open after cooling (opening 56b or 57b) on the right, on the left.

As long as the test liquid remains frozen in the upper subzone 52a, it does not pass through the openings 55b, 56b or 57b into the space 52b. In case of correct storage, the fill will appear as separate as before cooling and can be checked as desired at storage temperature. If the sample is thawed, the phase of the upper sub-space 52a becomes a liquid phase and permeates into the space 52b through the opening 55b, 56b or 57b. In this way, it is possible to ascertain whether the barrier 54 normally ruptured at storage temperature and thus whether the system operated in the desired manner.

According to the variants shown in FIGS. 1 to 5 , construction of more complex systems of these various elements and temperature recording or mixing of two or more liquid phases during heating is also possible.

6 shows a flow chart of the temperature monitoring method of a cryopreserved biological sample of the present invention. In the first step, a temperature monitoring device, for example 10, 20, 30 or 40 is prepared. In this case, an appropriate liquid or liquid mixture may be selected as the labeling material 7 according to the critical temperature value estimated to be monitored during refrigerated storage.

Depending on the appropriate liquid and the mixture ratio of the liquid, the melting point of the liquid can be determined to a desired value in the range of -20 to -140 ℃.

For example, Figure 7A shows the melting point profile as a function of the mixing ratio of alcohol and water, where a moderate increase in viscosity occurs as the temperature decreases in the range of 0 °C to -118 °C. If it is to be monitored with a critical temperature of -118°C, the ethanol percentage can be determined to be 93.5%. A slightly lower melting point than -60 °C can be achieved by adding potassium hydroxide (KOH) to the water, which is shown in Figure 7B based on the melting diagram. A mixture of water and antifreeze may also be used as a labeling material, as shown in the diagram of FIG. 8A. The table of FIG. 8B lists the freezing/melting points of pure liquids that may be used alone or mixed with other liquids suitable for use as labeling materials, including chloroform/cyclohexane mixtures or other miscible liquids, which are The mixture of solvents in Figure 9 can also refer to a matrix.

Liquid and plastic materials with good wettability at low temperatures and low viscosities are preferentially selected in order to minimize the widest possible displacement of position and further segregation.

If some critical temperature values to be monitored during frozen storage are proposed or if the temperature intervals that the sample must reach are to be precisely limited, several different labels, each with a different melting point, are placed in different chambers on or in the sample container. and can be used

As described above as an example in conjunction with the drawings herein, the label material may be transferred to the frozen state with or separately from the device depending on the embodiment of the device.

In the second step of the present invention, the instrument with the frozen sample in the receiving space of the sample container is stored at a storage temperature lower than the melting point.

Then, at any point during the cryopreservation, it can be checked by the labeling material whether unwanted heating has occurred, even if only temporarily, of the cryopreservation sample (step 3). At some point later, it is checked whether the label material is in the second sub-zone of the chamber. If this has occurred, it can be concluded that the melting point of the labeling substance has been exceeded and therefore the critical temperature to be monitored has been exceeded, even if it has only occurred for a very short time.

A suitable control process is described below for a temperature monitoring device comprising a covering or separation element as a barrier which ruptures as a result of heat shrinkage and forms an opening for allowing the liquid label material to penetrate into the second sub-zone of the chamber.

The following control process makes it possible to check the functionality of the instrument for each sample, in particular the functionality of the chamber acting as a labeling instrument.

In the first step, a labeling device in the form of a chamber equipped with a barrier at room temperature is checked before freezing the sample. The label material is located on one side of the barrier in a liquid state, for example in the first sub-zone of the chamber, the barrier is intact, and the label material is not present in the second sub-zone located below it. If this is not the case, the chamber is already damaged and cannot be used. If the chamber is integrated with the cover, it is no longer used and must be replaced with another one.

After cooling to a temperature below the melting point of the label material, the barrier is opened at a predetermined point by thermal contraction. However, since the labeled material is frozen, it retains its corresponding form prior to cooling. This can be confirmed in the second step. If this is not the case, a new cover must be used.

In the deep cooling state, it can be confirmed whether or not a temperature rise above the melting point of the labeling material has occurred at any desired time point. It should have flowed through the barrier into the second subzone. If the labeling material contains a dye as a labeling additive, it can be clearly identified in the third step.

If someone removes for use a deep-cooled sample that did not exceed the melting point of the labeling material, the state of the chamber will have the same appearance as in the first and second steps described above, i.e., any labeling material is located in the second sub-zone of the chamber. won't Nevertheless, exceeding the melting point of the labeling material is not indicated if the separating element is not torn and thus the barrier does not become permeable. This can be confirmed by leaving the chamber for a certain period of time after thawing. For example, if the chamber is integrated with a cover, only the cover can be left alone while the sample is subjected to other processes. If the separation element is properly ruptured and placed at a temperature below its melting point the entire time, the label liquid at room temperature will flow through the barrier into the test chamber, i.e., the second subzone. In this way, it is evident that the functional capability of the labeling device is demonstrated and that the storage temperature has met the requirements over the entire time period.

The control process described above is such that a barrier is disposed on a liquid absorbent material and the device is cooled to a storage temperature below the melting point of the labeling material to form a first covering impervious to the labeling material to a second covering permeable to the labeling material. It can function in a similar way even if it is formed by a covering that is irreversibly converted to a state.

10 shows a plurality of cross-sectional views of another embodiment 100 of a temperature monitoring mechanism of the present invention. Figure 10A shows an exploded cross-sectional view of the cover 101 of cryogenic tube 1 into which a device for temperature monitoring is integrated. Device 100 includes a cover 101 containing a cryogenic tube 1 having a receiving compartment 2 for receiving a biological sample 6 as well as a chamber containing a labeling material 7 separated from an absorbent material 103 by a barrier in an inactive state. Cover 101 is also referred to below as a temperature sensitive (T-sensitive) cover. Fig. 10B shows the cover mounted, and C shows the cryogenic tube 1 equipped with the T-sensitive cover 101 and the receiving chamber 2 for accommodating the elmological sample 6. 10A-C show the device 100 in an unactivated state.

The T-sensitive cover 101 has four parts: a screw insert 104, also called a screw-in part, and a liquid absorbent structure, also called an absorbent material, which absorbs the cover material 7, here a dyed liquid phase in a non-frozen state. An insert 103 embodied in the form of a plastic cushion containing a label material and a base 106 for screwing onto a commercially available cryogenic tube 1, here a base made in the form of a plastic cap 106.

In the device 100 in the inactive state, the plastic cushion 105 forms an impermeable cover to the labeling material, forming a barrier in the inactive state to prevent the labeling material 7 from contacting the absorbent material 103. This embodiment using a plastic cushion can be manufactured at a very low unit cost.

The screw-in part 104 is made transparent or translucent at at least one point so that at least a portion of the absorbent material 103 is visible through the screw-in part. For this purpose, the screw-in part 104 can be made of a transparent or translucent material, for example. Moreover, for this purpose, base 106 may be made of a transparent or translucent material. In this way, it is possible to confirm by simple visual inspection whether or not the color state of the absorbent material 103 has changed from above. For example, this means that the dyed labeling material 7 penetrates the absorbent material 103 and results in dyeing of the absorbent material, which will be described below.

The base or plastic cap 106 has an H-shaped cross-section, resulting in the formation of two cylindrical cavities. The upper cavity 102 forms a first sub-zone 102a in which the container 105 with the label material 7 is located and a second sub-zone 102b in which the absorbent material 103 is located.

For positive closure, a lower cavity is provided to receive the upper end of the cryogenic tube 1. The cryogenic tube 1 is sealed with a sealing ring 107. Positioned in the screw-in part 103 is a coupling portion 4, by which the screw-in part 104 can be rotated to the base 106 via, for example, a hexagonal hole. The screw-in part 104 may also include a blade 109 through which the screw-in part can rotate. To engage the screw, an external thread 104a is provided on the screw-in part 104, which external thread 104a engages a corresponding internal thread 106 of the base 106 provided on the side wall of the upper cavity 102.

As noted above, FIGS. 10A-C show the instrument 100 in an inactive state. This is because the plastic cushion 105 filled with the dyed labeling material is not yet broken and is impermeable, so that the liquid labeling material cannot flow out. The label material 7 is initially placed inside the plastic cushion 105 in liquid form. The absorbent material 103 placed thereon does not come into contact with the cover material 7 resulting in an intact plastic cushion 105. The labeling material may be formed of, for example, one of the above materials or a mixture of these materials. For example, the labeling material 7 can be dyed red by including a dye such as rhodamine B as a labeling additive.

The screw-in part 104 is initially half screwed (inactive). To turn it further, for example a quarter to a half turn, the plastic lock mechanism in threads 103a, 106a must be broken. This confirms that the present q-named temperature monitoring device and/or T-sensitive cover cannot be activated prior to use. However, prior to frozen storage of Apparatus 100 or Sample 6, this causes the absorbent material 103 located at the base to turn red and consequently unusable. Transport and intermediate storage of the cover 101 in an inactive state can be performed at any point in time, which is advantageous for transaction and inventory management.

10D shows the activation (transition to active mode) of the device after reaching the storage temperature, eg below -140°C.

Label material 7 is no longer liquid in vessel 105 at storage temperature and has already solidified. Activation of the device (activation mode switching) is performed by tightening the screw-in part when the label material is frozen. The screw-in part 104 includes protrusions 108 protruding from its lower surface facing the absorbent material 103, for example in the form of tips or spines. By tightening the screw-in part 104, the protrusion 108 punctures into the plastic cushion 105 and breaks. As a result, the absorbent material 103 does not come into direct contact with the red labeled material 7. At storage temperature, the labeling material is not absorbed into the absorbent material 103 because it exhibits high viscosity or is in a solid state. The ongoing thermal shock is not sufficient for diffusion.

The temperature monitoring mechanism is activated. As soon as the conversion temperature, which indicates the melting point of the mixture of labeling substances 7 during frozen storage, is exceeded, the frozen labeling substance 7 becomes liquid. As the temperature rises, the viscosity decreases until, at the critical temperature, the absorbent material 103 absorbs the liquid through capillary forces and acquires its color. This process is irreversible, that is, even if the labeling material 7 is re-frozen, the red dyeing of the absorbent material 103 is maintained. If it is confirmed through visual inspection that the absorbent material 103 is stained red, it can be concluded that the melting point of the labeling material and a temperature slightly higher than that are exceeded. In this case, the viscosity of the labeling material 7 is lowered so that the labeling material is sucked into the absorbent material 103 through capillary force. The type and thickness of the absorbent material 103 determines how quickly appreciable staining occurs. The absorbent material 103 may be, for example, filter paper, such as common kitchen towels or tobacco paper, compacts, cellulose discs, tissue cellulose discs, plaster and/or chalk dust.

What applies to the absorbent material 103 and the labeling material 7 inside the base 106 also applies to the temperature of the biosample 6. A red absorbent material indicates that biosample 6 has at least temporarily exceeded the aforementioned temperature. Since this process is not reversible even when the deep cooling is resumed, device 1 retains the information about non-applied heating as it is.

11 shows a plurality of cross-sectional views of another embodiment 110 of a temperature monitoring mechanism. The device 110 includes a cryogenic tube 1 having a receiving space 2 for receiving a biological sample 6 and a cover 111 . The temperature-sensitive cover includes four parts: a screw insert 114, also called a screw-in part, an insert 113 with a liquid-absorbing structure, also called an absorbent material, which must be capable of sucking up the liquid label material 7, and a label A container 105 for substance 7 and a base 116 for bonding onto a commercially available cryogenic tube 1, e.g. here embodied in a plastic cap.

11A shows cover 111 in an inactivated state prior to loading into cryogenic tube 1. 11B shows an exploded view of the individual parts of the cover 111. 11C shows instrument 110 in an activated state.

Unlike the embodiment shown in FIG. 10 , here the barrier is not in the form of a plastic cushion, but in the form of a glass ball 115 containing a label material 7 . To fill the glass ball 115 with the cover material 7 , the glass ball may include an opening 115a closed by the cover 119 after filling the glass ball 115 with the cover material 7 . Alternatively, the opening 115a may be bonded with a two-component adhesive or closed by welding with cooling of the labeling material 7. The absorbent material 113 is placed over the glass ball 115 closed in this way and, in this state (inactive state), does not come into contact with the label material 7.

To activate device 110 or switch to active mode, it is initially cooled to the storage temperature of the frozen storage at which label 7 is frozen. Then, in a manner similar to that described for the embodiment of FIG. 10 , screw-in part 114 is rotated into plastic cap 116 until the barrier separating label material 7 and absorbent material 113 is broken. In the case of the embodiment shown in FIG. 11 , the protrusion 118 of the screw-in part protrudes toward the glass ball 115 by the rotation of the screw-in part 114 and breaks the glass ball 115 . As shown in Figure 11C, this activates or puts the instrument 110 into an active state. However, labeling material 7 is not absorbed into the absorbent material 103 because it is very viscous or solid at storage temperature. The thermal shock that is still in progress is not sufficient for diffusion. During frozen storage, the frozen labeling substance 7 becomes liquid when the transition temperature, for example, the melting point of the mixture of labeling substances is continuously exceeded. As the temperature rises at the critical temperature, the viscosity decreases until the critical point is exceeded so that the absorbent material 113 at least partially absorbs the liquid labeling material 7 through capillary forces and exhibits its color.

An advantage of embodiments of the present invention using glass ball 115 as a barrier is that activation of the balloon simultaneously generates an audible feedback signal indicating that the balloon has now switched to active mode by generating a popping sound while breaking the ball.

12 shows an embodiment of a temperature monitoring mechanism 120 in multiple degrees. 12A shows a cross-sectional view of the entire cryogenic tube prior to activation of device 120 (inactive state). 12 A1 shows a bottom view of cryogenic tube 120. 12B also shows an exploded view to show the individual parts of the instrument 120. 12C shows a cross-sectional view of instrument 120 in an active state (active mode).

The device 120 includes a cryogenic tube having a receiving space 2 filled with a biological sample 6 therein and a screw-on cover 103 embodied in a conventional manner and which may have a fitting 4 . The appliance also includes a temperature monitoring device similar to that described in FIG. 10 or 11 for use in the screw cover. A feature of this embodiment is that the temperature monitoring device is integrated into the lower part 121 of the cryogenic tube 1, ie the end of the cryogenic tube opposite the cover. In contrast to the screw cover variant, the advantage of this embodiment is that there is no possibility of simple replacement of the temperature monitoring device since the biological sample 6 is located in the lower part in a frozen state and will be removed together.

A temperature monitoring device integrated with the lower part of the cryogenic tube consists of an absorbent material 123, a labeling material 7 which is initially stored in a liquid state in a closed oval container 105 made of plastic or glass, and which can be pushed up once and is normally placed in the lower part thereof. A circular part 124 with a 2D barcode 126 printed therein includes an opening 27 located therein and includes a plastic base 125 located at the base of the cryogenic tube 1 . On the opposite side not facing the bar code, part 124 includes a conical wedge 128 in the form of a cylinder. Part 124 is also referred to as tappet 124 below. Tappet 124 and preferably plastic base 125 are also made of a transparent or translucent material. The labeling material 7 may contain a dye such as rhodamine B as a labeling additive, and is dyed red.

In the manufacturing process, the parts are mounted as follows: A container 105 with label material 7 is inserted into a depression 122 at the bottom of the tube. As can be seen from the top of FIG. 12, the depression 122 has a curved shape on its upper side to fit the label container 105. An absorbent material 123 is placed thereon. Depression 122 thus forms a chamber for receiving label material container 105 and absorbent material 123 . The plastic base 125 is now put in place and is preferably fixedly connected to the cryogenic tube 1 in a non-detachable manner, for example by welding or gluing.

A tappet 124 with a barcode 126 is pressed in so that the wedge 128 penetrates precisely into the absorbent material 123 but not into the label container 105. For example, as shown in FIG. 12A, this can be accomplished by pressing the cryogenic tube 1 vertically onto the tappet 124 until it ends up flush with the base surface of the plastic base 125. In this case, too, there are two mounted parts: cover 3 and associated cryogenic tube 1, tappet 124, label material container 105, absorbent material 123, plastic base 125 and lower part 1, 121 including bartoad 126. . In this state, the base temperature monitoring device is not activated (inactive).

In use, the user fills lower part 1 with biosample 6, cools the tube to the cooling temperature, then applies pressure on the tappet 124 to the stop line 129 (indicated by the arrow in FIG. 12C) by forcing the base upwards. .

As a result, the container 105 of the frozen labeling liquid 7 is destroyed and the absorbent material 123 is pushed onto the container. The conical tappet 124 opens the opening and moves through it. There may optionally be a coating on the tappet 124, which results in a gas impermeable closure. Device 120 is now activated.

Label material 7 is no longer liquid at the storage temperature in container 105 and is already solid, or at least very viscous or solid so that it is not absorbed into absorbent material 123. The ongoing thermal shock is not sufficient for diffusion. As soon as the transition temperature indicating the melting point of the label material during frozen storage is exceeded, the frozen label material is liquefied. As the temperature rises, the viscosity decreases until a critical point is exceeded so that the absorbent material 123 can at least partially absorb the liquid labeling material 7 through capillary forces and exhibit its color.

If the first or second threshold temperature to be monitored is not exceeded, the base of the cryogenic tube remains undiscolored (eg, white as shown in Figure C1). This is shown in FIG. 12 C1 showing the bottom of the cryogenic tube 120 in a state in which the label material is not absorbed into the absorbent material. However, if during storage of the activated device 120 the second threshold temperature is not exceeded, the absorbent material 123 is filled and the base 121 of the cryogenic tube, in particular the tappet 124, is revealed to be dyed, eg dyed red. . This is shown in Figure 12C2, which shows the bottom of the cryogenic tube 120 with the label material absorbed into the absorbent material. This embodiment has the advantage that such barcode tubes are already widely available, and therefore the staining at the base can be easily read if barcode verification has been performed.

One embodiment of the present invention described above should not be construed as limiting the technical spirit of the present invention. The protection scope of the present invention is limited only by the matters described in the claims, and those skilled in the art can improve and change the technical spirit of the present invention in various forms. Therefore, such improvements and changes will fall within the protection scope of the present invention as long as they are obvious to those skilled in the art.

Claims (43)

a) a sample container having an accommodation space for accommodating a biological sample, and
b) a device for temperature monitoring of a cryopreserved biological sample comprising at least one chamber whose inner space is not fluidly connected to the containing space and which is partially filled with a labeling material whose melting point is in the range of -20 to -140°C. ,
the chamber having a barrier that causes the labeling material to flow in a delayed manner to the second subzone of the chamber if the labeling material is located in the first subzone of the chamber in a liquid state;
The mechanism is modified i); The label material is stored in a container enclosing the label material in a liquid state so as not to leak; The device is movably guided with respect to the container and is movable from a starting position to an energized position, wherein movement into the energized position causes the activating part to break the container at least at one point as a result of mechanical pressure, indicating that the container is in a liquid state. contains an active part that renders it permeable to a substance, or
variant ii); The barrier is a separating element permeable to the liquid labeling material and disposed between the first subzone and the second subzone, wherein when the device is cooled to a storage temperature below the melting point of the labeling material, the separating element will, as a result of heat shrinkage, at least A device for temperature monitoring of a cryopreserved biological sample, characterized in that it is formed to be ruptured at one point so that a liquid labeling substance can flow from a first sub-zone to a second sub-zone through an opening formed by at least one rupture point. According to claim 1,
Device according to variant i), characterized in that the barrier is disposed in the second sub-zone and is adjacent to the first sub-zone and is made of a material having a liquid absorbent structure. According to claim 1,
The apparatus according to variant i), characterized in that the container is embodied in a plastic cushion or a glass ball. According to claim 1,
The instrument of claim 1 , wherein the sample container includes a cover for closing the receiving space, and at least one chamber is integrated with the cover. According to claim 4,
The cover includes a base having an H-shaped cross section that can be press-fitted and/or screwed onto a sample container, the base having a container with a label material therein to form at least one chamber and a liquid absorbent structure. A device comprising a groove in which a material having a material is disposed, wherein the active part on the base is movably guided in the direction of the container. According to claim 4,
The cover includes a shaft coupled to an upper end of the accommodation space, and at least one chamber is integrated with the shaft. According to claim 1,
At least one chamber of the device is integral with the base of the sample container. According to claim 7,
The base of the sample container of modification i) includes a container having a labeling material therein and a groove in which a material having a liquid absorbent structure is disposed to form at least one chamber,
wherein the groove is closed by a base into which the activating part is movably guided. According to claim 8,
a) the activating position is fixed by means of a stop formed by the base, up to which the activating part can be pressed into the base; and/or
b) a device, characterized in that the base is fixedly connected to the base section of the sample container. According to claim 1,
The wall of the second sub-zone and/or the wall of the first sub-zone of the chamber
a) includes a scale indicating the period above the level or melting point of the labeled substance in each subzone; and/or
b) a device characterized in that it is transparent or translucent at least at one point. According to claim 10,
A device characterized in that the structure and/or components of the material having the liquid-absorbent structure are formed so that the diffusion rate of the labeling material inside the material decreases nonlinearly as the distance from the first sub-zone increases. According to claim 1,
Device according to variant ii), characterized in that the separating element permeable to the labeling substance is a porous separating wall, membrane, film, skin or capillary system. According to claim 12,
The device of claim 1 , wherein the separation element comprises at least one predetermined point of rupture at which it ruptures during cooling from the storage temperature of the device. According to claim 1,
A device characterized in that in the second sub-zone of variant ii) there is a substance with a melting point lower than a) gas or b) the labeled substance. According to claim 1,
The apparatus of claim 1 , wherein the outer wall of the chamber includes a closable opening in the first sub-area to fill the first sub-area with a label material. According to claim 1,
a) at least one chamber is formed by a container; and
b) An instrument further comprising a receiving portion coupled to an outer wall of the sample container. According to claim 1,
a) the sample container is a cryogenic tube;
b) at least one chamber is formed by a double-walled push-on part pressable on the outer wall surface of the cryogenic tube and coupled therearound in a push-on state. According to claim 17,
The apparatus, characterized in that the sample container is glued, melted or otherwise secured to the push-on part. According to claim 1,
The sample container is a bag for storing blood samples or stem cells held in a cassette, and at least one chamber includes:
a) joined to the outside of the bag;
b) exist to float freely inside the bag, or
c) a device characterized in that it is formed by a container coupled to a cassette. According to claim 1.
The labeling substances are octane-1ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, and butane-2. -A device comprising at least one alcohol selected from the group consisting of -ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, and benzyl alcohol, and at least one dye. According to claim 20,
The apparatus, characterized in that the dye is selected from the group comprising triphenylmethane dyes, rhodamine dyes, phenazine and phenothiazine dyes and azo dyes. According to claim 20,
The labeling substances are octane-1ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, and butane-2. -ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, at least two alcohols selected from the group consisting of benzyl alcohol and/or oil red, methyl red, brilliant grill, rhodamine B, neutral An instrument comprising at least one dye selected from the group consisting of red, methylene blue, or dyes used for cell staining in cytology. a) providing a temperature monitoring device according to claim 1 comprising at least one label material in a frozen state in a first sub-zone of the chamber, wherein the receiving space contains a cryopreserved sample;
b) cold storage of the appliance for cryopreservation; and
c) a method for monitoring the temperature of a cryopreserved sample, including the step of checking whether a labeling substance is located in the second sub-zone of the chamber at a later time point. According to claim 23,
A method according to claim 1 , wherein a parameter is determined to indicate a measurement of the amount of labeling substance that migrated to the second subzone of the chamber and/or located in the first subzone. According to claim 23,
A method characterized in that a melting point corresponding to a predetermined critical temperature to be monitored for exceeding or a viscosity of the labeling material melted at the critical temperature is selected as the labeling material. According to claim 23,
a) provided the temperature monitoring device comprises a covering, a cover and a base or isolating element;
b) to verify the functionality of the temperature monitoring device;
b1) checking whether there is no label material in the second sub-zone of at least one chamber after filling the first sub-zone with the label material and before freezing the label material;
b2) confirming whether there is no label material in the second sub-zone of the at least one chamber after freezing the labeling material located in the first sub-zone of the at least one chamber and before freezing storage of the appliance for cryopreservation; or
b3) if the cryopreserved sample is removed for use and there is no labeling material in the second subzone of the chamber, check whether the covering has correctly switched to the permeable second state or if at least one of the separation elements of the chamber has been correctly A method according to claim 1 , wherein at least one of the steps of determining whether rupture is performed. According to claim 23,
Octan-1ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butan-1,3-diol, butan-2-ol, pentane -1,5-diol, pentan-1-ol, cyclopentanol, benzyl alcohol and at least one alcohol selected from the group consisting of benzyl alcohol and at least one dye. The method of claim 27,
The method of claim 1, wherein the dye is selected from the group comprising triphenylmethane dyes, rhodamine dyes, phenazine and phenothiazine dyes, and azo dyes. The method of claim 27,
The labeling substances are octane-1ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, and butane-2. -ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, at least two alcohols selected from the group consisting of benzyl alcohol and/or oil red, methyl red, brilliant grill, rhodamine B, neutral A method comprising at least one dye selected from the group consisting of red, methylene blue, and dyes used for cell staining in cytology. According to claim 1,
wherein the activating part breaks the container at least at one point as a result of mechanical pressure, making the container permeable to the labeling substance in liquid state. According to claim 5,
The device, characterized in that the active part on the base is a screw-on part and is guided movably in the direction of the container. According to claim 8,
The device according to claim 1 , wherein the base has a mechanically and/or optoelectronically readable code. 33. The method of claim 32,
Wherein the mechanically and/or optoelectronically readable code is a barcode or 2D code. According to claim 9,
The device, characterized in that the base is fixed to the base region of the sample container by gluing, melting, welding or other fixing means. According to claim 16,
The device according to claim 1 , wherein the receptacle is an insertion pocket or sleeve into which a vessel can be inserted and/or into which a vessel can be inserted to stay on the sample vessel. According to claim 21,
Device characterized in that the rhodamine dye contains xanthine (xanthene). According to claim 28,
The method characterized in that the rhodamine dye contains xanthine (xanthene).

delete delete delete delete delete delete

Images